Three-dimensional Expansion Of Mouse Embryonic Stem Cells On Biomaterials

Embryonic stem cells (ESCs) have unlimited proliferation potential and capability todifferentiate into all cell and tissue types, and thus are ideal cell sources for tissue engineeringand cell therapy. ESCs can also be used for high-throughput drug screening and to transmitand express specific genes for therapeutic purposes. However,current in vitro cell culturemethod grows ES cells on the tissue-flask can not supply enough cells and it has become ahuge bottleneck in the practical application of stem cells.In this study the ESCs depends onthe feeder cells is used as the experimental material.Research targets: PET was used as scaffold material to support three dimensionalcultures of mESCs,to evaluate the amplification effect of static culture and dynamicculture;Compare the biocompatibility of PET,BioNOC,II,FibraCel Disks to mESCs/GFP,It isground work for further research of large-scale culture system in the bioreactor.Experiment methods:In this study,the mouse embryonic fibroblasts(MEF) were isolatedfrom ICR fetal mice.when the cells were passaged3or5times,they were inactivated intofeeder cells to culture mESCs/GFP.Alkaline phosphatase(ALP) staining and embryoid bodyformation assay were used to identify the pluripotency of mESCs/GFP;the mESCs/GFP wereinoculated into the PET material cultured by80%MEF-CM contioning103U LIF duringstatic and dynamic culture.Lactic acid and glucose concentrations in the culture media weremeasured to explore the metabolic characteristics of mESCs/GFP on PET. ALP staining andOct-4immunofluorescence staining were used to evluate the pluripotency of the cells.Scanning electron microscopy was used to detect distribution, morphology,and adhesion ofmESCs/GFP on PET.The mESCs/GFP were inoculated into PET,BioNOC â…¡,FibraCel Disksduring static culture.ALP staining and Oct-4immunofluorescence staining were used toevluate the pluripotency of the cells. Scanning electron microscopy was used to detectdistribution, morphology,and adhesion of mESCs/GFP on the biomaterials.Results:The MEF separated by our laboratory can be uesd as the feeder cells to culturethe undifferentiated mESCs/GFP;After cultured12days on PET by static and dynamicculture,mESCs/GFP were both ALP staining and Oct-4immunofluorescence stainingpositive,a high cell expansion fold and specific growth rate were obtained with spinner flaskversus static culture（43.75±0.64VS19.63±0.89å'Œ0.315±0.037VS0.248±0.009）, so themESCs/GFP inoculated into the PET material cultured in the spinner flask at80rpm can harvest moreundifferentiated cell;Compared to PET,BioNOCâ…¡ and FibraCel Disks can not only maintain the pluripotency of mESCs/GFP,and can also produed more cells,thus they have greatpotential to culture mESCs/GFP in the bioreactor.